This invention relates to derivatives of polymers which are useful as covalent chromatographic matrices. In one of its more particular aspects this invention relates to the preparation of such derivatives.
The need for purifying various biologically active materials in a facile manner has long been appreciated. Early methods of enzyme purification, for example, were cumbersome and time consuming. Recently it has been found that enzymes and other biologically active materials can be purified by a process which involves immobilization of the enzyme or other biologically active material which will be referred to herein as a ligand, followed by separation of the immobilized ligand from the mixture in which it is present. The ligand can then be used in its immobilized form, if desired, or can be released from the carrier on which it is immobilized by suitable chemical treatment and used in its non-immobilized form. The discovery of methods for covalently bonding ligands to polymeric carriers has advanced the practice of enzymology, immunology, and various other biological techniques.
One of the first methods for immobilizing biological ligands involved treatment of a polymer containing hydroxyl groups with an activating agent such as cyanogen bromide, CNBr. The activated polymer could then be used to directly bind various biological ligands to the polymer by means of covalent bonds. Porath et al. describe several chemical activation methods including the CNBr method in Porath, et al., "Immobilized Enzymes. Methods in Enzymology," K. Mosbach, Ed., Vol. 44, p. 19-45, Academic Press, New York (1976). Most of the early methods for activating polymers containing hydroxyl groups were subject to certain disadvantages which made their widespread use impractical. In particular CNBr activation procedures suffer from the following disadvantages: (1) the linkages formed between CNBr-activated hydroxyl containing polymers and the amino groups of ligands which are reacted with the activated polymers are labile; (2) the reaction between the activated polymer and ligand frequently results in the introduction of charged species which interfere with utilization of the reaction product in affinity absorption; (3) CNBr is a noxious, lachrimatory and poisonous chemical which requires special care in its handling.
Efforts to find another method other than the CNBr method for coupling ligands to hydroxyl containing polymers resulted in the use of a number of different reagents including triazine trichloride, N-hydroxy succinimide, 1,1'-carbonyldiimidazole and epoxy compounds. The use of epoxy compounds is described in Axen et al. Acta Chem. Scand B29: 471-474 (1975). Epichlorohydrin or 1,4-bis(2,3-epoxypropoxy) butane reacts with a hydroxyl group of an agarose gel to form an epoxide gel. The epoxide gel is then reacted with sodium thiosulfate to give a thiosulfate ester gel, which is then reduced by dithiothreitol to give a modified agarose gel containing a thiol group. This so-called thiol gel is then converted to a 2-pyridyl disulfide gel by means of 2,2'-dipyridyl disulfide. A solution of urease is then passed through a column of the disulfide gel to obtain an enzyme conjugate of high protein content and high catalytic activity. One disadvantage of this procedure is that the epoxy substituted polymer is not stable enough to store.
More recently the use of various organic sulfonates has found wide use in preparing immobilized affinity ligands. For example, Nilsson et al., Eur. J. Biochem., 112: 397-402 (1980) describes the coupling of a number of biomolecules to agarose gels by means of p-toluenesulfonyl chloride. The biomolecules used include nucleic acids and enzymes.
The use of other organic sulfonyl halides and the use of other hydroxyl group carrying supports are described in Nilsson et al., Biochem. Biophys. Res. Comm., 102: 449-457 (1981). The most active sulfonyl halide appears to be 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). Other hydroxyl group carrying supports mentioned in this reference are cellulose, diol-silica, glycophase-glass, and hydroxyethyl methacrylate.
U.S. Pat. No. 4,415,665 to Mosbach et al. teaches a method of covalently binding a biologically active substance containing amino, thiol or aromatic hydroxyl groups directly to a polymeric substance containing at least one hydroxyl group by forming a reactive sulfonate derivative of the polymeric substance and then reacting the thus activated polymeric substance directly with the biologically active organic substance. Although the use of sulfonyl halides has proven to be advantageous in many ways, the cost of the more active organic sulfonyl halides tends to be prohibitive and tresyl chloride, being a liquid, is less convenient to handle.
The purification of enzymes using consecutive thiol-disulfide interchange reactions is described in Carlsson et al., Acta Chem. Scand., B30: 180-182 (1976) in a communication in which urease is covalently bonded to agarose-2-pyridyl disulfide. Although this procedure is effective in carrying out the covalent chromatographic purification of urease, the preparation of the agarose-2-pyridyl disulfide involves a combination of steps utilizing an unstable epoxide derivative.
Mukaiyama et al. disclose the use of 1-methyl-2-alkoxypyridinium salts as reagents for preparing various 2-pyridyl sulfides. Mukaiyama et al., Chem. Lett., 1159-1162 (1975).
Hojo et al. successfully demonstrated the conversion of various alcohols to the corresponding thioalcohol by reacting the alcohol with 1-methyl-2-fluoropyridinium salts and sodium N,N-dimethyldithiocarbamate followed by reductive cleavage. The alcohols exemplified by these authors, including carbohydrates and steroids, were low molecular weight monomeric alcohols. Chem. Lett., 437-440 (1977).
A convenient method has now been found for preparing covalent chromatographic matrices utilizing a hydroxyl containing polymer which has been activated by reaction with 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP). The preparation and use of the activated hydroxyl containing polymer in forming covalent bonds with various ligands containing amino and sulfhydryl groups has been described in my copending application Ser. No. 679,525 filed of even date herewith, now U.S. Pat. No. 4,582,875, the disclosure of which is incorporated herein by reference. However, the covalently bound ligands are difficult to remove from the polymeric matrix. Therefore, it was found desirable to bind the ligand to the polymer in a manner such that the ligand could be readily removed when desired. The procedure involves conversion of the activated polymer to a thiol gel, that is, a sulfhydryl group containing polymer. The thiol gel can then be reacted with 2,2'-dipyridyl disulfide to form a 2-pyridyl disulfide derivative of the polymer. Thiol-disulfide interchange with the sulfhydryl group containing ligand causes the ligand to be linked to the polymer by means of a disulfide linkage. Removal of the ligand when desired can be readily accomplished by reduction of the disulfide linkage with a thiol such as dithiothreitol.
Two different routes to the thiol gel are available. In one, sodium dimethyl dithiocarbamate is used to convert the activated polymer to the corresponding dimethyl dithiocarbamyl derivative, which, by means of reductive cleavage is converted to the desired sulfhydryl substituted polymer, hereinafter referred to as the DS-gel.
Another route to a thiol gel involves treatment of the FMP activated polymer with dithiothreitol to form a dithiothreityl gel, a thiol gel, hereinafter referred to as the DTT-gel, in which the free sulfhydryl group is 4 carbon atoms removed from a thioether linkage to the polymer.
Depending upon the particular application for which the covalent chromatographic matrix is intended, either the DS-gel or the DTT-gel may be ideally suited for the particular chromatographic procedure which is to be carried out. For example, the DTT-gel may be particularly adapted for use in those instances where the ligand contains bulky groups which might prevent it from approaching close enough to the polymer to attack the disulfide linkage, were it not for the space provided between the polymer surface and the disulfide linkage by the intervening 4 carbon atom chain.